We are using two approaches to identify and isolate genes involved in the regulated growth and differentiation of the mammalian embryo and fetus. The first of these is insertional mutagenesis, in which mutant phenotypes are generated by the insertion of exogenous DNA into a gene. This also creates a molecular tag at the mutant locus, providing direct access to the gene. For several years, we have analyzed transgenic mouse strains derived from embryonic stem (ES) cells deliberately infected in tissue culture with retroviruses to generate insertional mutations. We have screened over 40 proviral insertions specifically for mutations that lead to prenatal lethality, indicating that the gene is absolutely essential for normal development. This screen has identified several recessive prenatal lethal mutations tightly linked to, and presumably caused by, proviral insertion. For the molecular analysis of two of these, the 412-k and the 412-r mutations, we isolated the retroviral insertion sites and approximately 100 kilobases of surrounding DNA on overlapping cosmids. Fragments of candidate genes were obtained using exon-trapping and extended using 5' and 3' RACE. The expression of these candidate genes during normal embryonic development was studied using RT-PCR and Northern blotting. For 412-r, the best candidate is a gene that appears to have multiple splicing forms and for which one non- coding exon is the site for retroviral integration. We are currently isolating corresponding cDNAs and mapping the transcribed regions to determine the complete structure of the gene. Also, we are addressing whether and how expression of this gene is affected in homozygous 412-r mutant embryos. To accomplish this, we developed methods to accurately genotype individual embryos using PCR. Similar approaches are being applied to the 412-k mutation. Phenotypic analysis of both mutants has been complicated by the presence of an unlinked early embryonic mutation in the stock strain. We have recently begun breeding both mutations into a different stock. The second approach to screen for developmental genes is based on the premise that many critical regulatory molecules are ubiquitinated during embryonic development, and can be identified by their interaction with ubiquitinating enzymes. For this screen we utilized one of the few well characterized ubiquitin ligases, Nedd-4. We first determined that Nedd-4 is broadly expressed during early embryonic development and showed that by midgestation, expression is most pronounced in the limb and tail buds; regions undergoing proliferation and remodeling. We therefore did a yeast two-hybrid screen on a cDNA library from midgestation mouse embryos and isolated 3 novel genes. For two of them, human homologues have been described from an EST screening study performed by another laboratory. We have shown that two of the three clones can be ubiquitinated in vitro, lending further support to the idea that theses are substrates for Nedd-4. Expression analysis for one of them has been completed and has revealed a pattern that is quite similar to that of Nedd-4. We currently are determining the expression patterns of the other genes.